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Article

Phenolic Compounds in Edible Tropaeolum majus L. Leaves and Its In Vitro Digestion

by
Ivana Vrca
1,*,
Dora Jukić
1,
Josip Radić
2 and
Ivana Anđelić
1
1
Faculty of Science, University of Split, Ruđera Boškovića 33, 21000 Split, Croatia
2
Faculty of Chemistry and Technology, University of Split, Ruđera Boškovića 35, 21000 Split, Croatia
*
Author to whom correspondence should be addressed.
Analytica 2025, 6(2), 14; https://doi.org/10.3390/analytica6020014 (registering DOI)
Submission received: 21 February 2025 / Revised: 11 April 2025 / Accepted: 14 April 2025 / Published: 18 April 2025
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Abstract

:
Tropaeolum majus L. is an edible plant known for its therapeutic and medicinal effects, as it possesses bioactive compounds (polyphenols, glucosinolates, fatty acids) and has various biological activities, which makes it interesting and makes it the research objective of this work. The aim of this study was to extract the phenolic compounds present in the T. majus plant by maceration and ultrasound-assisted extraction techniques using two solvents: 80% ethanol and water. In vitro digestion was performed to see how stable the phenolic components are after digestion. An LC-MS/MS instrument was used to identify and quantify the phenolic components. The highest extraction yield for the T. majus extract was obtained when 80% ethanol was used as the solvent after ultrasound-assisted extraction (32.63 ± 2.28 mg/0.5 g fresh material), while the opposite was true for the T. majus extract when water was used as the solvent and maceration as the technique (21.00 ± 3.26 mg/0.5 g fresh material). However, water extracted more phenolic components for identification. In general, the major compounds before in vitro digestion with commercial enzymes and with 80% ethanol and water as the solvents were p-hydroxybenzoic acid, protocatechuic acid, p-coumaric acid, caffeic acid and chlorogenic acid. After in vitro digestion using both solvents and extraction procedures, the stable phenolic compounds were p-hydroxybenzoic acid (>67%) and p-coumaric acid (>35%). Caffeic acid and quinic acid were not detected after digestion. The stability of certain phenolic components could influence the use of such extracts as dietary supplements with beneficial effects on human health, making them interesting for the general population.

1. Introduction

Tropaeolum majus L. (garden nasturtium) is a plant that belongs to the Tropaeolaceae family and is known for its therapeutic and medicinal effects [1]. It is considered an extremely important and valuable edible plant due to the polyphenols it contains, as well as its content of the glucosinolate glucotropaeolin and of fatty acids [1,2]. It is interesting to note that T. majus is considered an unconventional food plant, and it has different uses depending on the country [3]. Unconventional food plants (UFPs) are generally native, naturalized or exotic species whose leaves, roots, flowers or stems are edible but are not normally used in the human diet [4]. The leaves, flowers and immature green seeds of T. majus are edible and widely used in traditional medicine for the treatment of various diseases, such as urinary tract infections, constipation, asthma and cardiovascular diseases, as well as an antibacterial, antiseptic, diuretic, laxative and stimulant [5,6,7,8,9]. Binet [10], Goos et al. [11], Barboza et al. [12] and Gasparoto et al. [13] reported on the diuretic effects of T. majus extracts in vitro and/or in vivo. Vrca et al. [1] reported on the strong antimicrobial and antiproliferative activities of benzyl isothiocyanate contained in T. majus essential oil. Benzyl isothiocyanate also showed significant stability after in vitro digestion using commercially available enzymes [14]. Garzón and Wrolstad [15] reported on the antioxidant activity of the orange flowers of T. majus, which were able to scavenge the radicals ABTS and DPPH. In Europe, T. majus is cultivated as an ornamental plant due to its beautiful flowers and leaves, which are also used in medicine [16]. All parts of the plant are mainly used fresh. The leaves are round, with soft edges, yellow-green veins and long petioles that become entangled in the supports [17,18]. The chemical profile of T. majus is not fully known. Considering the available literature, it can be assumed that the chemical composition of T. majus varies depending on the plant part, cultivation method and location, as well as the color of the flowers, which are the best studied so far [5,15,19,20,21]. Salem et al. [22] reported that the influence of the geographical location where the plants are grown has different effects on their chemical profile and biological activity.
The extraction of the desired bioactive components is the first and most important step in experimental research. Extraction procedures play a major role in the efficiency of extraction and the stability of the desired components [23]. Maceration is a simple and inexpensive extraction technique with low implementation costs, especially for small-scale laboratory extraction [24]. The ultrasound-assisted extraction (UAE) technique uses mechanical, thermal and cavitation effects to extract bioactive compounds. During exposure to ultrasound, the cell wall is destroyed, while the release and diffusion of components in the cell are accelerated [25,26]. The desired compounds can be extracted from fresh, dried or frozen plant material [27]. Chromatographic methods are mainly used for the identification and quantification of phenolic components [28,29].
The phenolic components contained in plants have high antioxidant activity and their consumption as functional foods may have a positive effect on human health [30]. To explore the effects of food composition on human health, digestion in the upper gastrointestinal tract is increasingly being simulated [31].
Static in vitro digestion models usually involve three phases (the oral, gastric and intestinal phases) to determine the bioaccessibility of the compounds of interest after digestion [31,32,33]. The benefits of phenolic compounds for human health are assessed by analyzing their bioaccessibility (the fraction released from the food matrix into the gastrointestinal tract that becomes available for absorption in the small intestine) [34]. During the digestion process, some phenolic compounds may be transformed into other compounds that have a different bioaccessibility and bioactivity profile or that are not released from the food matrix at all, resulting in weaker beneficial effects on human health [35].
Therefore, the objectives of this research were as follows: (1) the extraction of bioactive compounds from fresh T. majus leaves by maceration extraction and UAE techniques; (2) the in vitro digestion of T. majus extracts with commercially available enzymes; and (3) the identification of their chemical composition by LC-MS/MS analysis before and after in vitro digestion, as well as the investigation of the bioactive compounds’ bioaccessibility after in vitro digestion with a view to determining their possible future application in the food or pharmaceutical industry.

2. Materials and Methods

2.1. Plant Material

The T. majus plant was grown from seed (Royal Seeds, HortuSi s.r.l., Longiano (FC), Italy) in a thermoregulation room under conditions of 12 h day/12 h night at a temperature of 24 °C (Figure 1). Vegetative and edible parts of the plant (leaves) were harvested after three weeks. Fresh T. majus leaves were cut into small pieces and the extraction of bioactive compounds was performed using maceration and ultrasound-assisted extraction techniques.

2.2. Maceration and Ultrasonic-Assisted Extraction Techniques

Freshly cut T. majus leaves (0.5 g) were extracted by maceration with 25 mL of two solvents (80% ethanol and water) at room temperature for three days (72 h) with occasional stirring. The UAE method was also used to extract phenolic constituents from the leaves of the T. majus plant at a temperature of 25 °C for a period of 30 min at 37 kHz (ratio 1:50) according to method described in Žitek et al. [36]. Subsequently, the extracts obtained were centrifuged at 2000× g for 5 min and filtered through a blue ribbon filter paper (LabExpert, Kefo d.o.o., Ljubljana, Slovenia). Subsequently, the extracts were crude-evaporated at 50 °C using a Büchi Rotavapor R-200 (Flawil, Switzerland) to remove ethanol, while the aqueous extracts were freeze-dried using an Alpha 1–4 LSCplus freeze dryer (Osterode am Harz, Germany). The obtained dry material was then dissolved in ultrapure water (Milli-Q water) at a stock concentration of 10 mg/mL to test in vitro digestion.

2.3. In Vitro Digestion

For the simulation of in vitro digestion, salivary amylase, pancreatin and bile salts were obtained and used from Sigma-Aldrich, Merck KgaA, St. Louis, MO, USA. Rabbit gastric extract (RGE15) was purchased from Lipolytech (Marseille, France).
Electrolyte stock solutions were used for the preparation of digestive fluids (simulated saliva, simulated gastric and intestinal fluids) according to the method described in detail in Brodkorb et al. [31]. In vitro digestion was performed according to the method described in detail by Vrca et al. [33]. The oral phase involved the dilution of the T. majus extracts at a 1:1 ratio (v/v) with a simulated salivary fluid containing salivary amylase (15 U/mL) for 2 min at a temperature of 37.0 °C and a pH 7 (V = 625 µL). The oral bolus was then diluted 1:1 with simulated gastric fluid (SGF) containing pepsin and gastric lipase (2000 U/mL, 60 U/mL) and incubated with agitation at a pH 2 for 1 h at a temperature of 37.0 °C (V = 1.25 mL). At the end of in vitro digestion, the gastric mucus was diluted 1:1 with simulated intestinal fluid (SIF). Pancreatin and bile salts were added to the SIF and incubated for 2 h at a temperature of 37 °C and a pH 7.0 (trypsin activity 100 U/mL, bile salts 10 mM) (Figure 2). The final concentration of T. majus extracts was 1 mg/mL in a final volume of 2.5 mL. After digestion, all samples were briefly placed on ice to prevent the enzymatic reaction. The digested samples were then centrifuged at 2000× g for 5 min at room temperature. All digestion experiments were performed in triplicate (n = 3). The bioaccessibility of the main compounds in the extracts corresponds to the ratio of their concentrations in the supernatants (extracts after digestion) and the concentrations of phenolic compounds in the extracts before in vitro digestion.

2.4. Reagents and Standards for LC/MS-MS Analysis

For LC-MS/MS analysis, HPLC-grade acetonitrile was purchased from Merck KgA (Darmstadt, Germany). Formic acid was purchased from Prolabo (VWR, International, Radnor, PA, USA). The pure phenolic compounds (standards) listed in Table 1 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.5. Chemical Characterization of Phenolic Compounds

For liquid chromatography–mass spectrometry (LC-MS/MS) analysis, the T. majus extracts were prepared according to method described in detail by Vrca et al. [37] and Živković et al. [38]. A SCIEX—Triple TOF 6600+ mass spectrometer coupled to a liquid chromatography system (SCIEX—ExionLC) with a binary pump, as well as a Phenomenex Kinetex Core–Shell 2.6 µm C18 100 Å, 100 × 2.1 mm column, thermostated at 40 °C, were used for analysis. The solvents used were 0.1% formic acid in water (A) and acetonitrile (B). The flow rate was 0.3 mL/min, while the injection volume was 10 µL. The ion spray voltage was set at −4500 V in the negative mode. The compounds of interest contained in the sample extracts were characterized by mass spectra and retention times determined using commercial standards. For the quantitative analysis of phenolic compounds, a calibration curve was established by injecting known concentrations (0.3 ng/mL–2 µg/mL) of pure compounds. Quantification was performed using Sciex OS 1.6.1.29803 software and was based on the MS/MS peak area of the extracted ion chromatograms of selected characteristic fragments for each compound, except chlorogenic acid, for which the TOF MS chromatogram of the precursor was used for quantification. The reference standards with their precursor ions and the fragments used for quantification and their retention times are listed in Table 1. All samples were run in triplicate, including the standards. The quantities of phenolic compounds were calculated using calibration curves, and the results are expressed in µg/g dry weight (DW).

2.6. Statistical Analyses

GraphPad Prism version 9 (GraphPad Software, Inc., San Diego, CA, USA) was used for the statistical analyses. An unpaired t-test was used to examine the difference between each extraction technique and solvent in terms of the extraction yield obtained. A two-way ANOVA test was used for statistical data processing. Then, Sidak’s multiple comparison test was used to examine the difference between the individual components present in the same species extracted with the same extraction technique and solvent before and after in vitro digestion (undigested and digested samples) (different letters a–b).

3. Results and Discussion

Phenolic compounds were extracted from the edible leaves of T. majus using maceration extraction (72 h at room temperature) and UAE (30 min, 25 °C, 37 kHz) with water and 80% ethanol as the solvents. Extractions were performed in triplicate (n = 3). The results are expressed in mg/0.5 g of fresh T. majus plant material (Table 1) depending on the solvents used for each extraction technique (maceration and ultrasound assisted extraction, UAE). The highest extraction yield for the T. majus extract was obtained when 80% ethanol was used as the solvent after UAE, while the opposite was true for the T. majus extract when water was used as the solvent and maceration as the technique. Based on the extraction yield results, UAE is a better extraction technique and 80% EtOH is a better solvent (Table 2).
When the maceration technique was used, with water as the solvent, the main constituents of the extract of T. majus were p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, syringic acid, p-coumaric acid, o-coumaric acid, caffeic acid, ferulic acid and chlorogenic acid (Table 3). p-Hydroxybenzoic acid, o-coumaric acid and ferulic acid are stable after in vitro digestion, whereas p-coumaric acid and chlorogenic acid show significant degradation (decrease by 64.61% and 65.06%, respectively). When 80% ethanol was used in the maceration technique, vanillic acid, syringic acid, o-coumaric acid and ferulic acid were not detected, but quinic acid and quercetin were. After digestion, protocatechuic acid, caffeic acid, quinic acid and quercetin were no longer detected. p-Coumaric acid and chlorogenic acid also showed significant degradation when 80% ethanol was used as a solvent (decrease of 33.03% and 97.74%). The major constituents in the undigested extracts of T. majus after UAE with water used as a solvent were p-hydroxybenzoic acid, protocatechuic acid, p-coumaric acid and chlorogenic acid (Table 3). When 80% ethanol was used as a solvent, caffeic acid and quinic acid were also detected and quantified. The main constituents in the digested samples after UAE were p-hydroxybenzoic acid, p-coumaric acid and chlorogenic acid; this was the case for both solvents. Protocatechuic acid, caffeic acid and quinic acid, which were present in the water extracts after UAE, were no longer detected after digestion. Chlorogenic acid showed the strongest degradation after digestion with both methods and solvents (Table 3).
Phenolic compounds were extracted from T. majus leaves using the maceration extraction and UAE techniques. The maceration technique was used as a reference method in comparison to UAE, an advanced extraction technique. Modern extraction methods for plant metabolites, such as UAE, show no deterioration in the content of bioactive compounds in the treated foods and are characterized by non-invasive temperatures [39]. According to Dzah et al. [40], in the extraction of polyphenols with UAE, it is noteworthy that the optimal efficiency is observed when using a lower frequency, namely below 40 kHz, which is consistent with our method. The physical ability of ultrasonic waves allows for the disruption or deformation of cell walls, resulting in a better efficiency in the extraction of the desired bioactive components, which in turn leads to better yields compared to traditional techniques [26,41], which is also consistent with our results. Maceration, as the most commonly used conventional extraction technique, has advantages such as simplicity and low cost, but it also has disadvantages such as long extraction times, large amounts of solvent, and lower yields [23], which is in line with our results.
Due to its high content of biologically active compounds, T. majus is a valuable component of the diet and can serve as a raw material for the production of nutraceuticals [42]. Bazylko et al. [16,42] reported the scavenging activity of aqueous and hydroethanolic extracts from a garden nasturtium herb obtained in Poland on cinnamoylquinic acids, primarily chlorogenic acid and vitamin C. According to our results, chlorogenic acid is one of the most important acids in the leaves of T. majus (with the highest concentration seen when 80% ethanol was used as a solvent). As far as the leaves and stems of nasturtium are concerned, there are still few data on their nutritional properties in the literature [3]. In general, its aqueous and hydroethanolic extracts are mainly used in medicine and cosmetics. Bazylko et al. [42] reported that nasturtium leaf extract helps in the treatment of urinary tract infections and has antihypertensive and anticoagulant effects. According to Ercan and Doğru [43], the extraction method and solvent affect the content of phenolic components in the extract, which can also vary depending on the environment in which the plant grows, the freshness of the plant, the drying method and storage conditions. The results we have obtained are consistent with these observations. Boo [44] reported the antioxidant activity of p-coumaric acid in reducing oxidative stress and inflammatory reactions. According to Ercan and Doğru [43], the main components in various extracts of Nasturtium officinale (watercress, order Brassicales) were quinic acid, foumaric acid, protocatechuic acid, p-coumaric acid, ferulic acid and caffeic acid. Phenolic components are the main compounds in plant extracts that exhibit significant antioxidant activity, which directly correlates with the elimination of free radicals and superoxide [39]. Musolino et al. [45] reported not only on the antioxidant activity of T. majus extracts, but also on the potential use of these extracts as novel anti-arthritis and anti-obesity agents. Due to the biological activities that T. majus exhibits thanks to its phenolic components, it is of utmost importance to investigate their gastrointestinal stability. Mathew et al. [46] reported that the influence of substituents on the phenyl ring and conjugated carbon skeleton plays an important role in the antioxidant activity of phenolic compounds. Ferulic acid and caffeic acid were generally more efficient free radical scavengers than vanillic acid and protocatechuic acid [46].
In vitro digestion studies usually include simulating the conditions of the stomach and small intestine using enzymes and other digestive fluids [47,48].
Bioaccessibility indicates the amount of phenolic compounds absorbed by the epithelial layer of the gastrointestinal tract [49]. In this study, we determined the content of phenolic compounds before and after the oral, gastric and intestinal phases (three-phase in vitro digestion model) using LC/MS-MS.
The results obtained show that the concentration of some phenolic components was significantly reduced after the gastric and intestinal phases, while some phenolic acids could no longer be detected at all after in vitro digestion [30,50].
These results are also consistent with our results. After in vitro digestion, the following main components were detected: p-hydroxybenzoic acid, p-coumaric acid and chlorogenic acid. These results can be explained by the damage of some phenolic components, as the pH and other environmental conditions in the stomach and intestinal phase may not be suitable [30]. Higher concentrations of certain phenolic acids after in vitro digestion compared to undigested samples can be explained by the transition from an acidic gastric environment to a slightly alkaline intestinal environment, whereby certain compounds are released from the plant matrix and remain stable [51]. Subbiah et al. [48] reported that the fermentation phase in the colon can reduce the bioaccessibility of phenolic compounds, while the fermentation phases in the intestine and colon have the ability to metabolize new phenolic compounds [52]. No such results were obtained in this study. Therefore, the presence of phenolic compounds is influenced by their release from the food matrix, while their stability can be influenced by biochemical factors such as enzymes, bile salts and physicochemical factors such as pH, ionic strength or temperature [53].

4. Conclusions

Phenolic compounds were extracted from edible T. majus leaves using maceration for 72 h at room temperature and UAE, using water and 80% ethanol as solvents. After the use of UAE as the technique and 80% ethanol as the solvent, the extraction yield was highest. Generally, the main compounds before and after in vitro digestion using commercially available enzymes and using 80% ethanol as the solvent were p-hydroxybenzoic acid, protocatechuic and chlorogenic acids. p-hydroxybenzoic acid, protocatechuic acid, o-coumaric acid, ferulic acid, and chlorogenic acid were the main compounds before in vitro digestion and p-coumaric acid, o-coumaric acid and chlorogenic acid were the main compounds after digestion when water was used as the solvent. Water as a solvent proved to be better than 80% ethanol for the extraction of more phenolic components from fresh leaves of the T. majus plant. Some phenolic components were not detected after in vitro digestion due to unfavorable pH and environmental factors. After in vitro digestion, the stable phenolic compounds were p-hydroxybenzoic acid and p-coumaric acid using both solvents and extraction procedures, while caffeic acid and quinic acid were not detected after digestion. The bioaccessibility of certain phenolic components could influence the use of these extracts as dietary supplements with positive effects on human health. Therefore, future research will continue in this direction.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/analytica6020014/s1, Figure S1: Chromatogram of T. majus extract after maceration using EtOH as solvent; Figure S2: Chromatogram of T. majus extract after UAE using EtOH as solvent.

Author Contributions

Conceptualization, I.V.; methodology, I.V., D.J., I.A., and J.R.; validation, I.V. and I.A.; formal analysis, I.V., D.J., I.A. and J.R.; investigation, I.V. and D.J.; resources, I.V. and J.R.; data curation, I.V. and I.A.; writing—original draft preparation, I.V.; writing—review and editing, I.V., D.J., I.A. and J.R.; visualization, I.V.; supervision, I.V.; project administration, I.V.; funding acquisition, I.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the institutional project “Chemical analysis, in vitro gastrointestinal stability and biological activity of bioactive compounds of traditional and aromatic plants for the purpose of their application in the industry”, a project funded by the Faculty of Science in Split through the 2023–2025 Research Fund.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.

Acknowledgments

A mass spectrometer (SCIEX—Triple TOF 6600+) coupled to a liquid chromatography system (SCIEX—ExionLC) with a binary pump was procured as part of the project “Functional integration of the University of Split through the development of scientific research infrastructure in the building of the three faculties”, funded by the European Commission through the European Regional Development Fund.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Vrca, I.; Ramić, D.; Fredotović, Ž.; Smole Možina, S.; Blažević, I.; Bilušić, T. Chemical Composition and Biological Activity of Essential Oil and Extract from the Seeds of Tropaeolum majus L. Var. Altum. Food Technol. Biotechnol. 2022, 60, 533–542. [Google Scholar] [CrossRef]
  2. Brondani, J.C.; Cuelho, C.H.F.; Marangoni, L.D.; de Lima, R.; Guex, C.G.; Bonilha, I.d.F.; Manfron, M.P. Traditional Usages, Botany, Phytochemistry, Biological Activity and Toxicology of Tropaeolum majus L.—A Review. Bol. Latinoam. Y Del Caribe Plantas Med. Y Aromat. 2016, 15, 264–273. [Google Scholar]
  3. Cobus, D.; Nunes, G.; Maior, L.d.O.; Lacerda, L.G.; Ito, V. Unconventional Food Plants (UFPs): An Approach to the Nutritional and Functional Properties of Nasturtium (Tropaeolum majus L.). Food Sci. Today 2023, 1, 1–6. [Google Scholar] [CrossRef]
  4. Bass, A.; Theodoro, H.; Minello, L.V.; Scur, L.; Pansera, M.R.; Sartori, V.C. Plantas Alimentícias Não Convencionais—PANC: Resgatando a Soberania Alimentar e Nutricional; Editora da Universidade de Caxias do Sul: Los Angeles, CA, USA, 2020; ISBN 978-85-7061-992-1. [Google Scholar]
  5. Garzón, G.A.; Manns, D.C.; Riedl, K.; Schwartz, S.J.; Padilla-Zakour, O. Identification of Phenolic Compounds in Petals of Nasturtium Flowers (Tropaeolum majus) by High-Performance Liquid Chromatography Coupled to Mass Spectrometry and Determination of Oxygen Radical Absorbance Capacity (ORAC). J. Agric. Food Chem. 2015, 63, 1803–1811. [Google Scholar] [CrossRef]
  6. Lorenzi, H.; Matos, F.J.d.A. Plantas Medicinais No Brasil: Nativas e Exóticas; Instituto Plantarum de Estudos da Flora: Nova Odessa, Brazil, 2002; ISBN 8586714186. [Google Scholar]
  7. Ferreira, R.B.G.; Vieira, M.C.; Zárete, N.A.H. Análise de Crescimento de Tropaeolum majus “Jewel” Em Função de Espaçamentos Entre Plantas. Rev. Bras. Plantas Med. 2005, 7, 57–66. [Google Scholar]
  8. Ferro, D. Fitoterapia: Conceitos Clínicos; Atheneu: São Paulo, Brazil, 2008; ISBN 978857379824. [Google Scholar]
  9. Bown, D. Encyclopedia of Herbs and Their Uses; DK Publishing: London, UK, 1995; ISBN 0888503342. [Google Scholar]
  10. Binet, L. A Biologist Physician in the Country. Biol. Med. 1964, 53, 5–28. [Google Scholar]
  11. Goos, K.-H.; Albrecht, U.; Schneider, B. Wirksamkeit Und Verträglichkeit Eines Pflanzlichen Arzneimittels Mit Kapuzinerkressenkraut Und Meerrettich Bei Akuter Sinusitis, Akuter Bronchitis Und Akuter Blasenentzündung Im Vergleich Zu Anderen Therapien Unter Den Bedingungen Der Täglichen Praxis. Arzneimittelforschung 2011, 56, 249–257. [Google Scholar] [CrossRef]
  12. Barboza, L.N.; Prando, T.B.L.; Dalsenter, P.R.; Gasparotto, F.M.; Gasparotto, F.; Jacomassi, E.; Araújo, V.D.O.; Lourenço, E.L.B.; Gasparotto Junior, A. Prolonged Diuretic Activity and Calcium-Sparing Effect of Tropaeolum majus: Evidence in the Prevention of Osteoporosis. Evidence-Based Complement. Altern. Med. 2014, 2014, 958291. [Google Scholar] [CrossRef]
  13. Gasparotto Junior, A.; Gasparotto, F.M.; Boffo, M.A.; Lourenço, E.L.B.; Stefanello, M.É.A.; Salvador, M.J.; Da Silva-Santos, J.E.; Marques, M.C.A.; Kassuya, C.A.L. Diuretic and Potassium-Sparing Effect of Isoquercitrin—An Active Flavonoid of Tropaeolum majus L. J. Ethnopharmacol. 2011, 134, 210–215. [Google Scholar] [CrossRef]
  14. Vrca, I.; Burčul, F.; Blažević, I.; Bratanić, A.; Bilušić, T. Comparison of Gastrointestinal Stability of Isothiocyanates from Tropaeolum majus L. Altum Using in Vitro and Ex Vivo Digestion Methods. Croat. J. food Sci. Technol. 2021, 13, 160–166. [Google Scholar] [CrossRef]
  15. Garzón, G.A.; Wrolstad, R.E. Major Anthocyanins and Antioxidant Activity of Nasturtium Flowers (Tropaeolum majus). Food Chem. 2009, 114, 44–49. [Google Scholar] [CrossRef]
  16. Bazylko, A.; Parzonko, A.; Jeż, W.; Osińska, E.; Kiss, A.K. Inhibition of ROS Production, Photoprotection, and Total Phenolic, Flavonoids and Ascorbic Acid Content of Fresh Herb Juice and Extracts from the Leaves and Flowers of Tropaeolum majus. Ind. Crops Prod. 2014, 55, 19–24. [Google Scholar] [CrossRef]
  17. Das, M.; Biswas, S.K.; Zaman, S.; Mitra, A. Nasturtium (Tropaeolum majus) an Annual Herb Has Medicinal Property to Cure Throat Sore and It Has Antivirus Property. Int. J. Environ. Agric. Sci. 2019, 3, 16. [Google Scholar]
  18. Jakubczyk, K.; Janda, K.; Watychowicz, K.; Łukasiak, J.; Wolska, J. Garden Nasturtium (Tropaeolum majus L.)—a Source of Mineral Elements and Bioactive Compounds. Rocz. Panstw. Zakl. Hig. 2018, 69, 119–126. [Google Scholar]
  19. Navarro-González, I.; González-Barrio, R.; García-Valverde, V.; Bautista-Ortín, A.; Periago, M. Nutritional Composition and Antioxidant Capacity in Edible Flowers: Characterisation of Phenolic Compounds by HPLC-DAD-ESI/MSn. Int. J. Mol. Sci. 2014, 16, 805–822. [Google Scholar] [CrossRef]
  20. Niizu, P.Y.; Rodriguez-Amaya, D.B. Flowers and Leaves of Tropaeolum majus L. as Rich Sources of Lutein. J. Food Sci. 2006, 70, S605–S609. [Google Scholar] [CrossRef]
  21. Rop, O.; Mlcek, J.; Jurikova, T.; Neugebauerova, J.; Vabkova, J. Edible Flowers—A New Promising Source of Mineral Elements in Human Nutrition. Molecules 2012, 17, 6672–6683. [Google Scholar] [CrossRef]
  22. Salem, O.; Szwajkowska-Michałek, L.; Przybylska-Balcerek, A.; Szablewski, T.; Cegielska-Radziejewska, R.; Świerk, D.; Stuper-Szablewska, K. New Insights into Bioactive Compounds of Wild-Growing Medicinal Plants. Appl. Sci. 2023, 13, 13196. [Google Scholar] [CrossRef]
  23. Mathews, A.; Arbal, A.V.; Kaarunya, A.; Jha, P.K.; Le-Bail, A.; Rawson, A. Conventional vs. Modern Extraction Techniques in the Food Industry. In Extraction Processes in the Food Industry; Elsevier: Amsterdam, The Netherlands, 2024; pp. 97–146. ISBN 978-0-12-819516-1. [Google Scholar]
  24. Đurović, S.; Domínguez, R.; Pateiro, M.; Teslić, N.; Lorenzo, J.M.; Pavlić, B. Industrial Hemp Nutraceutical Processing and Technology. In Industrial Hemp; Elsevier: Amsterdam, The Netherlands, 2022; pp. 191–218. ISBN 978-0-323-90910-5. [Google Scholar]
  25. Chemat, F.; Rombaut, N.; Sicaire, A.-G.; Meullemiestre, A.; Fabiano-Tixier, A.-S.; Abert-Vian, M. Ultrasound Assisted Extraction of Food and Natural Products. Mechanisms, Techniques, Combinations, Protocols and Applications. A Review. Ultrason. Sonochem. 2017, 34, 540–560. [Google Scholar] [CrossRef]
  26. Shen, L.; Pang, S.; Zhong, M.; Sun, Y.; Qayum, A.; Liu, Y.; Rashid, A.; Xu, B.; Liang, Q.; Ma, H.; et al. A Comprehensive Review of Ultrasonic Assisted Extraction (UAE) for Bioactive Components: Principles, Advantages, Equipment, and Combined Technologies. Ultrason. Sonochem. 2023, 101, 106646. [Google Scholar] [CrossRef]
  27. Abubakar, A.R.; Haque, M. Preparation of Medicinal Plants: Basic Extraction and Fractionation Procedures for Experimental Purposes. J. Pharm. Bioallied Sci. 2020, 12, 1–10. [Google Scholar] [CrossRef] [PubMed]
  28. Bajkacz, S.; Baranowska, I.; Buszewski, B.; Kowalski, B.; Ligor, M. Determination of Flavonoids and Phenolic Acids in Plant Materials Using SLE-SPE-UHPLC-MS/MS Method. Food Anal. Methods 2018, 11, 3563–3575. [Google Scholar] [CrossRef]
  29. Yilmaz, M.A. Simultaneous Quantitative Screening of 53 Phytochemicals in 33 Species of Medicinal and Aromatic Plants: A Detailed, Robust and Comprehensive LC–MS/MS Method Validation. Ind. Crops Prod. 2020, 149, 112347. [Google Scholar] [CrossRef]
  30. Ozkan, G.; Sakarya, F.B.; Tas, D.; Yurt, B.; Ercisli, S.; Capanoglu, E. Effect of In Vitro Digestion on the Phenolic Content of Herbs Collected from Eastern Anatolia. ACS Omega 2023, 8, 12730–12738. [Google Scholar] [CrossRef]
  31. Brodkorb, A.; Egger, L.; Alminger, M.; Alvito, P.; Assunção, R.; Ballance, S.; Bohn, T.; Bourlieu-Lacanal, C.; Boutrou, R.; Carrière, F.; et al. INFOGEST Static in Vitro Simulation of Gastrointestinal Food Digestion. Nat. Protoc. 2019, 14, 991–1014. [Google Scholar] [CrossRef] [PubMed]
  32. Minekus, M.; Alminger, M.; Alvito, P.; Ballance, S.; Bohn, T.; Bourlieu, C.; Carrière, F.; Boutrou, R.; Corredig, M.; Dupont, D.; et al. A Standardised Static in Vitro Digestion Method Suitable for Food—An International Consensus. Food Funct. 2014, 5, 1113–1124. [Google Scholar] [CrossRef]
  33. Vrca, I.; Fredotović, Ž.; Jug, B.; Nazlić, M.; Dunkić, V.; Jug, D.; Radić, J.; Možina, S.S.; Restović, I. Chemical Profile of Kumquat (Citrus japonica Var. Margarita) Essential Oil, In Vitro Digestion, and Biological Activity. Foods 2024, 13, 3545. [Google Scholar] [CrossRef]
  34. Carbonell-Capella, J.M.; Buniowska, M.; Barba, F.J.; Esteve, M.J.; Frígola, A. Analytical Methods for Determining Bioavailability and Bioaccessibility of Bioactive Compounds from Fruits and Vegetables: A Review. Compr. Rev. Food Sci. Food Saf. 2014, 13, 155–171. [Google Scholar] [CrossRef]
  35. Rein, M.J.; Renouf, M.; Cruz-Hernandez, C.; Actis-Goretta, L.; Thakkar, S.K.; da Silva Pinto, M. Bioavailability of Bioactive Food Compounds: A Challenging Journey to Bioefficacy. Br. J. Clin. Pharmacol. 2013, 75, 588–602. [Google Scholar] [CrossRef]
  36. Žitek, T.; Kučuk, N.; Postružnik, V.; Leitgeb, M.; Knez, Ž.; Primožič, M.; Marevci, M.K. Synergistic Effect of Supercritical and Ultrasound-Assisted Ginger (Zingiber Officinale Roscoe) Extracts. Plants 2022, 11, 2872. [Google Scholar] [CrossRef]
  37. Vrca, I.; Orhanović, S.; Pezelj, I.; Sušić, K.; Dunkić, V.; Kremer, D.; Nazlić, M. Identification of Phenolic Compounds Present in Three Speedwell (Veronica L.) Species and Their Antioxidant Potential. Antioxidants 2024, 13, 738. [Google Scholar] [CrossRef]
  38. Živković, J.; Barreira, J.C.M.; Stojković, D.; Ćebović, T.; Santos-Buelga, C.; Maksimović, Z.; Ferreira, I.C.F.R. Phenolic Profile, Antibacterial, Antimutagenic and Antitumour Evaluation of Veronica Urticifolia Jacq. J. Funct. Foods 2014, 9, 192–201. [Google Scholar] [CrossRef]
  39. Gadjalova, A.V.; Mihaylova, D.S. Ultrasound-Assisted Extraction of Medicinal Plants and Evaluation of Their Biological Activity. Food Res. 2019, 3, 530–536. [Google Scholar] [CrossRef]
  40. Dzah, C.S.; Duan, Y.; Zhang, H.; Wen, C.; Zhang, J.; Chen, G.; Ma, H. The Effects of Ultrasound Assisted Extraction on Yield, Antioxidant, Anticancer and Antimicrobial Activity of Polyphenol Extracts: A Review. Food Biosci. 2020, 35, 100547. [Google Scholar] [CrossRef]
  41. Mnayer, D.; Fabiano-Tixier, A.-S.; Petitcolas, E.; Ruiz, K.; Hamieh, T.; Chemat, F. Extraction of Green Absolute from Thyme Using Ultrasound and Sunflower Oil. Resour. Technol. 2017, 3, 12–21. [Google Scholar] [CrossRef]
  42. Bazylko, A.; Granica, S.; Filipek, A.; Piwowarski, J.; Stefańska, J.; Osińska, E.; Kiss, A.K. Comparison of Antioxidant, Anti-Inflammatory, Antimicrobial Activity and Chemical Composition of Aqueous and Hydroethanolic Extracts of the Herb of Tropaeolum majus L. Ind. Crops Prod. 2013, 50, 88–94. [Google Scholar] [CrossRef]
  43. Ercan, L.; Doğru, M. Determination of Phenolic Compounds in Nasturtium Officinale by LC-MS / MS Using Different Extraction Methods and Different Solvents. Int. J. Chem. Technol. 2023, 7, 124–130. [Google Scholar] [CrossRef]
  44. Boo, Y.C. P-Coumaric Acid as An Active Ingredient in Cosmetics: A Review Focusing on Its Antimelanogenic Effects. Antioxidants 2019, 8, 275. [Google Scholar] [CrossRef]
  45. Musolino, V.; Marrelli, M.; Perri, M.R.; Palermo, M.; Gliozzi, M.; Mollace, V.; Conforti, F. Centranthus ruber (L.) DC. and Tropaeolum majus L.: Phytochemical Profile, In Vitro Anti-Denaturation Effects and Lipase Inhibitory Activity of Two Ornamental Plants Traditionally Used as Herbal Remedies. Molecules 2022, 28, 32. [Google Scholar] [CrossRef]
  46. Mathew, S.; Abraham, T.E.; Zakaria, Z.A. Reactivity of Phenolic Compounds towards Free Radicals under in Vitro Conditions. J. Food Sci. Technol. 2015, 52, 5790–5798. [Google Scholar] [CrossRef]
  47. Sensoy, I. A Review on the Food Digestion in the Digestive Tract and the Used in Vitro Models. Curr. Res. Food Sci. 2021, 4, 308–319. [Google Scholar] [CrossRef] [PubMed]
  48. Subbiah, V.; Ebrahimi, F.; Agar, O.T.; Dunshea, F.R.; Barrow, C.J.; Suleria, H.A.R. In Vitro Digestion and Colonic Fermentation of Phenolic Compounds and Their Antioxidant Potential in Australian Beach-Cast Seaweeds. Sci. Rep. 2024, 14, 4335. [Google Scholar] [CrossRef]
  49. Wu, H.; Liu, Z.; Lu, P.; Barrow, C.; Dunshea, F.R.; Suleria, H.A.R. Bioaccessibility and Bioactivities of Phenolic Compounds from Roasted Coffee Beans during in Vitro Digestion and Colonic Fermentation. Food Chem. 2022, 386, 132794. [Google Scholar] [CrossRef] [PubMed]
  50. Ozkan, G.; Franco, P.; De Marco, I.; Capanoglu, E.; Esatbeyoglu, T. Investigating the Effects of Supercritical Antisolvent Process and Food Models on Antioxidant Capacity, Bioaccessibility and Transepithelial Transport of Quercetin and Rutin. Food Funct. 2022, 13, 4469–4477. [Google Scholar] [CrossRef]
  51. de Paulo Farias, D.; de Araújo, F.F.; Neri-Numa, I.A.; Dias-Audibert, F.L.; Delafiori, J.; Catharino, R.R.; Pastore, G.M. Effect of in Vitro Digestion on the Bioaccessibility and Bioactivity of Phenolic Compounds in Fractions of Eugenia Pyriformis Fruit. Food Res. Int. 2021, 150, 110767. [Google Scholar] [CrossRef]
  52. Garzón, A.G.; Van de Velde, F.; Drago, S.R. Gastrointestinal and Colonic in Vitro Bioaccessibility of γ-Aminobutiric Acid (GABA) and Phenolic Compounds from Novel Fermented Sorghum Food. LWT 2020, 130, 109664. [Google Scholar] [CrossRef]
  53. Sęczyk, Ł.; Sugier, D.; Świeca, M.; Gawlik-Dziki, U. The Effect of in Vitro Digestion, Food Matrix, and Hydrothermal Treatment on the Potential Bioaccessibility of Selected Phenolic Compounds. Food Chem. 2021, 344, 128581. [Google Scholar] [CrossRef]
Analytica 06 00014 g001
Figure 1. Tropaeolum majus L. plant (Author: Ivana Vrca).
Figure 1. Tropaeolum majus L. plant (Author: Ivana Vrca).
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Figure 2. Flow diagram of in vitro digestion.
Figure 2. Flow diagram of in vitro digestion.
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Table 1. Precursor and quantitation fragments m/z and retention time of phenolic compounds [37].
Table 1. Precursor and quantitation fragments m/z and retention time of phenolic compounds [37].
CompoundPrecursor m/zFragment m/ztr (min)
p-hydroxybenzoic acid137.0293.033.48
protocatechuic acid153.02109.032.21
Gentisic acid153.02109.033.21
Vanillic acid167.03152.014.66
Gallic acid169.01125.021.33
Syringic acid197.04123.015.59
p-coumaric acid163.04119.057.18
o-coumaric acid163.04119.059.16
Caffeic acid179.03135.044.9
Ferulic acid193.05134.038.19
Chlorogenic acid353.08179,034.64
Quinic acid191.0585.034.64
Sinapic acid223.06193.018.49
Rosmarinic acid359.08161.029.88
Cinnamic acid147.05103.0611.17
Epicatechin289.07109.036.25
Catechin289.07109.034.17
Resveratrol227.07143.0510.42
Astringin405.12243.067.50
EGCG (Epigallocatechin gallate)457.08169.016.95
Hesperetin301.07164.0112.61
Quercetin301.03151.0011.36
Myricetin317.03151.0010.03
Apigenin269.04117.0312.46
Naringenin271.06151.0012.14
Rutin609.15300.038.86
Table 2. Extraction yield of Tropaeoulum majus L. extract using different extraction techniques and solvents.
Table 2. Extraction yield of Tropaeoulum majus L. extract using different extraction techniques and solvents.
ExtractsT. majus Extract After MacerationT. majus Extract After UAE
Extraction Yield (mg/0.5 g of fresh material)
80% EtOH extract25.97 ± 2.66 a32.63 ± 2.28 b
Water extract21.00 ± 3.26 a25.9 ± 3.84 a,b
Extractions were performed in triplicate (n = 3). Data are presented as mean ± SD, n = 3. An unpaired t test was used for statistical data processing to examine the difference between each extraction technique and different solvents (different letters a–b).
Table 3. Identification and quantification of the phenolic compounds in various T. majus extracts before and after in vitro digestion.
Table 3. Identification and quantification of the phenolic compounds in various T. majus extracts before and after in vitro digestion.
T. majus Extract After Maceration T. majus Extract After UAE
Compound80% EtOH
Extract UD		80% EtOH Extract DWater
Extract UD		Water
Extract D		80% EtOH Extract UD80% EtOH Extract DWater
Extract UD		Water
Extract D
(µg/g of FM)
p-hydroxybenzoic acid0.0091 ± 0.00009 a0.0088 ± 0.00002 a0.00773 ± 0.00005 a0.0073 ± 0.00011 a0.0110 ± 0.00004 a0.0074 ± 0.00627 a
0.0096 ± 0.00005 a0.0089 ± 0.00003 a
Protocatechuic acid0.0091 ± 0.00005 an.d.a0.00838 ± 0.00011 a0.0072 ± 0.00001 a0.0113 ± 0.00005 an.d. a0.0089 ± 0.00002 an.d. b
Vanillic acidn.d.n.d.0.0133 ± 0.00035 an.d. bn.d.n.d.n.d.n.d.
Syringic acidn.d.n.d.0.0101 ± 0.00004 an.d. bn.d.n.d.n.d.n.d.
p-coumaric acid0.0195 ± 0.00003 a0.01306 ± 0.00082 a0.0616 ± 0.00251 a0.0218 ± 0.00011 b0.0242 ± 0.00003 a0.0209 ± 0.01766 a0.0211 ± 0.00023 a0.0210 ± 0.00058 a
o-coumaric acidn.d.n.d.0.0089 ± 0.00012 a0.0089 ± 0.00002 an.d.n.d.n.d.n.d.
Caffeic acid0.0125 ± 0.00009 an.d. b0.0354 ± 0.00076 an.d. b0.0154 ± 0.00001 an.d. an.d.n.d.
Ferulic acidn.d.n.d.0.0114 ± 0.00014 a0.0104 ± 0.00029 an.d.n.d.n.d.n.d.
Chlorogenic acid0.6369 ± 0.00444 a0.01439 ± 0.00029 b0.0332 ± 0.00016 a0.0116 ± 0.00024 b0.5625 ± 0.06324 a0.0119 ± 0.01009 b0.0153 ± 0.00158 a0.0140 ± 0.00006 b
Quinic acid0.5696 ± 0.01557 an.d. bn.d.n.d.0.4599 ± 0.03130 an.d. bn.d.n.d.
Quercetin0.0220 ± 0.00094 an.d. bn.d.n.d.n.d.n.d.n.d.n.d.
Legend: n.d.—not detected; FM—fresh material; UD—undigested; D—digested; UAE—ultrasound assisted extraction. Data are presented as mean ± SD, n = 3. A two-way ANOVA test was used for statistical data processing. Then, Sidak’s multiple comparison test was used to examine the difference between the individual components present in the same species extracted with the same extraction technique and solvent before and after in vitro digestion (undigested and digested samples) (different letters a–b).
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MDPI and ACS Style

Vrca, I.; Jukić, D.; Radić, J.; Anđelić, I. Phenolic Compounds in Edible Tropaeolum majus L. Leaves and Its In Vitro Digestion. Analytica 2025, 6, 14. https://doi.org/10.3390/analytica6020014

AMA Style

Vrca I, Jukić D, Radić J, Anđelić I. Phenolic Compounds in Edible Tropaeolum majus L. Leaves and Its In Vitro Digestion. Analytica. 2025; 6(2):14. https://doi.org/10.3390/analytica6020014

Chicago/Turabian Style

Vrca, Ivana, Dora Jukić, Josip Radić, and Ivana Anđelić. 2025. "Phenolic Compounds in Edible Tropaeolum majus L. Leaves and Its In Vitro Digestion" Analytica 6, no. 2: 14. https://doi.org/10.3390/analytica6020014

APA Style

Vrca, I., Jukić, D., Radić, J., & Anđelić, I. (2025). Phenolic Compounds in Edible Tropaeolum majus L. Leaves and Its In Vitro Digestion. Analytica, 6(2), 14. https://doi.org/10.3390/analytica6020014

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